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Functional characterization of two 5-HT3 receptor splice variants isolated from a mouse hippocampal cell line

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  • Molecular and cellular physiology
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Abstract

Two splice variants of the ligand-gated 5-hydroxytryptamine or serotonin 5-HT3 receptor that differ in a six-amino-acid deletion were cloned by polymerase chain reaction from the hippocampus x neuroblastoma cell line HN9.10e. When expressed in Xenopus oocytes, both variants individually formed 5-HT3 receptors that revealed no significant differences in current responses to the agonists 5-HT and 1-phenylbiguanide and block by the specific antagonist LY-278,584-maleate. For both receptors, the monovalent cations Na+, K+, Rb+ and Li+ showed the same relative permeability; NH +4 permeated ≈ 2.7 times better than Na+, and Tris+ was only poorly permeable. In contrast to other reports, the receptors were completely and reversibly blocked by extracellular Cs+ in both oocytes and native HN9.10 cells. Moreover, Ca2+ was not permeant and exhibited a concentration-dependent decrease (0.9–18 mM) of the 5-HT-induced currents without affecting the inward rectification of the current/voltage relation. The two receptors were reversibly inhibited by nanomolar concentrations of the specific inhibitor of protein kinase C (PKC) bisindolylmale-imide, but not by the equipotent and less specific inhibitor staurosporine. A regulatory effect on both 5-HT3 receptor subunits by PKC-mediated protein phosphorylation might be possible, however, a functional role of the two splice variants present in one cell remains to be determined.

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References

  1. Arbuckle JB, Meigel I, Boddeke HWGM, Docherty RJ (1993) Inhibition of calcineurin reduces desensitization of 5-HT3 receptors in NG108-15 neuroblastoma × glioma hybrid cells. J Physiol (Lond) 473:267P

    Google Scholar 

  2. Derkach V, Surprenant A, North RA (1989) 5-HT3 receptors are membrane ion channels. Nature 339:706–709

    Article  PubMed  CAS  Google Scholar 

  3. Downie DL, Hope AG, Lambert JJ, Peters JA, Blackburn TP, Jones BJ (1994) Pharmacological characterization of the apparent splice variants of the murine 5-HT3R-A subunit expressed in Xenopus laevis oocytes. Neuropharmacology 33:473–482

    Article  PubMed  CAS  Google Scholar 

  4. Eckstein F (1985) Nucleoside phosphorothioates. Annu Rev Biochem 54:367–402

    Article  PubMed  CAS  Google Scholar 

  5. Eiselé JL, Bertrand S, Galzi JL, Devillers-Thiéry A, Changeux JP, Bertrand B (1993) Chimaeric nicotinic-serotonergic receptor combines distinct ligand binding and channel specificities. Nature 366:479–483

    Article  PubMed  Google Scholar 

  6. Gill CJ, Peters JA, Lambert JJ, Julius D (1993) Modulation by divalent cations of current responses mediated by a cloned murine 5-HT receptor (5-HT3R-A) expressed in HEK 293 cells. Br J Pharmacol 109:98P

    Google Scholar 

  7. Gilon P, Yakel JL (1995) Activation of 5-HT, receptors in Xenopus oocytes does not increase cytoplasmic Ca2+ levels. Receptors and Channels 3:83–88

    PubMed  CAS  Google Scholar 

  8. Goldin AL (1992) Maintenance of Xenopus laevis and oocyte injection. In: Rudy B, Iverson LE (eds) Methods in enzymology (207). Academic, San Diego, pp 266–279

    Google Scholar 

  9. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch 391:85–100

    Article  PubMed  CAS  Google Scholar 

  10. Hille B (1992) Ionic channels of excitable membranes. Sinauer Associates, Sunderland, Mass., pp 337–361

    Google Scholar 

  11. Hope AG, Downie DL, Sutherland L, Lambert JJ, Peters JA, Burchell B (1993) Cloning and functional expression of an apparent splice variant of the murine 5-HT3 receptor A sub-unit. Eur J Pharmacol 245:187–192

    Article  PubMed  CAS  Google Scholar 

  12. Hoyer D, Clarke DE, Fozard JR, Hartig PR, Martin GR, Mylecharane EJ, Saxena PR, Humphrey PPA (1994) International union of pharmacology classification of receptors for 5-hydrxytryptamine (serotonin). Pharmacol Rev 46: 157–203

    PubMed  CAS  Google Scholar 

  13. Hussy N, Lukas W, Jones KA (1994) Functional properties of a cloned 5-hydroxytryptamine ionotropic receptor subunit: comparison with native mouse receptors. J Physiol (Lond) 481: 311–323

    CAS  Google Scholar 

  14. Imoto K, Busch C, Sakmann B, Mishina M, Konno T, Nakai J, Bujo H, Mori Y, Fukuda K, Numa S (1988) Rings of negatively charged amino acids determine the acetylcholine receptor channel conductance. Nature 335:645–648

    Article  PubMed  CAS  Google Scholar 

  15. Lee HJ, Hammond DN, Large TH, Roback JD, Sim JA, Brown DA, Otten UH Wainer BH (1990) Neuronal properties and trophic activities of immortalized hippocampal cells from embryonic and young adult mice. J Neurosci 10: 1779–1787

    PubMed  CAS  Google Scholar 

  16. Maniatis T, Fritsch E, Sambrook J (1982) Molecular cloning. A laboratory manual. Cold Spring Habor Laboratory, Cold Spring Harbor, N.Y.

    Google Scholar 

  17. Maricq AV, Peterson AS, Brake AJ, Myers RM, Julius D (1991) Primary structure and functional expression of the 5-HT3receptor, a serotonin-gated ion channel. Science 254:432–437

    Article  PubMed  CAS  Google Scholar 

  18. Miquel MC, Emerit MB, Gozlan H, Hamon M (1991) Involvement of tryptophan residue(s) in the specific binding of agonists/antagonists to 5-HT3 receptors in NG108-15 clonal cells. Biochem Pharmacol 42:1453–1461

    Article  PubMed  CAS  Google Scholar 

  19. Miquel MC, Emerit MB, Gingrich JA, Nosjean A, Hamon M, El Mestikawy S (1995) Developmental changes in the differential expression of two serotonin 5-HT3 receptor splice variants in the rat. J Neurochem 65:475–483

    Article  PubMed  CAS  Google Scholar 

  20. Pearson RB, Kemp BE (1991) Protein kinase phosphorylation site sequences and consensus specificity motifs: tabulations. In: Hunter T, Sefton BM (eds) Methods in enzymology (200). Academic, San Diego, pp 62–81

    Google Scholar 

  21. Peters JA, Hales TG, Lambert JJ (1988) Divalent cations modulate 5-HT3 receptor-induced currents in N1E-115 neuroblastoma cells. Eur J Pharmacol 151:491–495

    Article  PubMed  CAS  Google Scholar 

  22. Robertson B, Bevan S (1991) Properties of 5-hydroxytryptamine 3 receptor-gated currents in adult rat dorsal root ganglion neurons. Br J Pharmacol 102:272–276

    PubMed  CAS  Google Scholar 

  23. Swope SL, Moss SJ, Blackstone CD, Huganir RL (1992) Phosphorylation of ligand-gated ion channels: a possible mode of synaptic plasticity. FASEB J 6:2514–2523

    PubMed  CAS  Google Scholar 

  24. Toullec D, Pianetti P, Coste H, Bellevergue P, Grand-Perret T, Ajakane M, Baudet V, Boissin P, Boursier E, Loriolle F, Duhamel L, Charon D, Kirilovsky J (1991) The bisindolyl- maleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J Biol Chem 24:15771–15781

    Google Scholar 

  25. Uetz P, Abdelatty F, Villarroel A, Rappold G, Weiss B, Koenen M (1994) Organisation of the murine 5-HT3 receptor gene and assignment to human chromosome 11. FEBS Lett 339: 302–306

    Article  PubMed  CAS  Google Scholar 

  26. Van Hooft, JA, Vijverberg PM (1995) Phosphorylation controls conductance of 5-HT, receptor ligand-gated ion channels. Receptors Channels 3:7–12

    PubMed  Google Scholar 

  27. Werner P, Kawashima E, Reid J, Hussy N, Lundström K, Buell G, Humbert Y, Jones KA (1994) Organization of the mouse 5-HT, receptor gene and functional expression of two splice variants. Mol Brain Res 26:233–241

    Article  PubMed  CAS  Google Scholar 

  28. Yakel JL (1992) 5-HT3receptors as cation channels. In: Hamon M (ed) Central and peripheral 5-HT, receptors. Academic, New York

    Google Scholar 

  29. Yakel JL, Jackson MB (1988) 5-HT, receptors mediate rapid responses in cultured hippocampus and a clonal cell line. Neuron 1:615–621

    Article  PubMed  CAS  Google Scholar 

  30. Yakel JL, Shao XM, Jackson MB (1990) The selectivity of the channel coupled to the 5-HT, receptor. Brain Res 533:46–52

    Article  PubMed  CAS  Google Scholar 

  31. Yakel JL, Shao XM, Jackson MB (1991) Activation and desensitization of the 5-HT,receptor in a rat glioma × mouse neuroblastoma hybrid cell. J Physiol (Lond) 436:293–308

    CAS  Google Scholar 

  32. Yang J (1990) Ion permeation through 5-hydroxytryptaminegated channels in neuroblastoma N18 cells. J Gen Physiol 96:1177–1198

    Article  PubMed  CAS  Google Scholar 

  33. Yang J, Mathie A, Hille B (1992) 5-HT3, receptor channels in dissociated rat superior cervical ganglion neurons. J Physiol (Lond) 448:237–256

    CAS  Google Scholar 

  34. Zhang 1, Oz M, Weight FF (1995) Potentiation of 5-HT, receptor-mediated responses by protein kinase C activation. Neuroreport 6:1336–1340

    Google Scholar 

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Authors and Affiliations

  1. Molecular Neurobiology of Signal Transduction, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, D-37077, Göttingen, Germany

    Maike Glitsch, Erhard Wischmeyer & Andreas Karschin

  2. Cellular Neurobiology, Max-Planck Institute for Biophysical Chemistry, D-37077, Göttingen, Germany

    Maike Glitsch

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  1. Maike Glitsch
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  2. Erhard Wischmeyer
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  3. Andreas Karschin
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Glitsch, M., Wischmeyer, E. & Karschin, A. Functional characterization of two 5-HT3 receptor splice variants isolated from a mouse hippocampal cell line. Pflügers Arch — Eur J Physiol 432, 134–143 (1996). https://doi.org/10.1007/s004240050115

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  • DOI: https://doi.org/10.1007/s004240050115

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